Inhibition of calcineurin by cyclosporine A exerts

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Inhibition of calcineurin by cyclosporine A exerts multiple effects on human melanoma cell lines HT168 and WM35 TAMÁS JUHÁSZ1, CSABA MATTA1, GÁBOR VERESS1, GEORGINA NAGY2, ZSOLT SZÍJGYÁRTÓ3, ZSANETT MOLNÁR3, JÁNOS FODOR4, RÓZA ZÁKÁNY1 and PÁL GERGELY3 1

Department of Anatomy, Histology and Embryology, 2Department of Dermatology, 3Cell Biology and Signalling Research Group of the Hungarian Academy of Sciences, Department of Medical Chemistry and 4Department of Physiology Medical and Health Science Centre, University of Debrecen, Hungary Received September 19, 2008; Accepted November 21, 2008 DOI: 10.3892/ijo_00000225

Abstract. The immunosuppressant cyclosporine A (CsA) is a specific pharmacological inhibitor of calcineurin, the Ca2+-calmodulin activated phospho-Ser/Thr-specific protein phosphatase. Although calcineurin-inhibiting compounds are applied for local treatment of psoriasis or atopic dermatitis in dermatological practice, little is known about the functions of calcineurin in epidermis-derived malignancies. We investigated the effects of CsA on two human melanoma cell lines, the metastasis forming HT168 and WM35 established from an RGP primary lesion. CsA of 2 μM lowered the enzyme activity by 50% and caused elevation in both mRNA and protein expression of calcineurin. Cell proliferation was diminished, as well as the cellular morphology and the actin organization were altered in both cell lines. CsA increased cell death moderately in both cell lines and reduced the metabolic activity of HT168 cells, but not that of WM35 cells. CsA also elevated the expressions of both Bcl-2 and ERK1/2. Fibronectin guided migration of HT168 cells was stimulated under the effect of CsA, while that of WM35 cells was reduced, moreover, HT168 cells switched from the expression of ß3 to ß1 integrin, but WM35 cells continued to express ß3. Based on our results we propose a multiple, partly malignancy-dependent role of calcineurin in these melanoma cell lines. Introduction Calcineurin or PP2B is a Ca-calmodulin activated phosphoSer/Thr-specific protein phosphatase, which was first detected in brain and skeletal muscle (1), and is best known as a key regulator of T-lymphocyte activation via dephosphorylation

_________________________________________ Correspondence to: Dr Pál Gergely, Department of Medical Chemistry, Medical and Health Science Centre, University of Debrecen, Nagyerdei krt. 98, H-4032 Debrecen, Hungary E-mail: [email protected]

Key words: calcineurin, cyclosporine A, migration, actin organization, integrins, apoptosis, ERK1/2, Bcl-2

and consequent nuclear translocation of the transcription factor: Nuclear Factor of Activated T-lymphocytes (NFAT) (2,3). Pharmacological inhibitors of calcineurin, such as cyclosporine A (CsA), tacrolimus or pimecrolimus recently became popular in the dermatological practice for topic treatment of diseases of skin which accompany activation of T-lymphocytes, e.g. atopic dermatitis (4). Local application of these compounds have no significant side effects (5), but it is reported that long-term systemic application of calcineurin inhibitors as immunosuppressants following organ transplantations may increase the incidence of skin tumours, particularly squamous cell carcinoma (6). Some physiological roles of calcineurin played in keratinocytes have been reported recently. It serves as a factor in the regulation of the terminal differentiation of keratinocytes (7) and it modulates the DNA repair following UV irradiation (8). Application of calcineurin inhibitors for the treatment of hypopigmentary skin disorders has also been suggested (9), but the investigation of the physiological role of calcineurin in the biology of melanocytes, the pigment producing cells of epidermis, has just started (10). Melanocytes develop from neural crest during embryonic life and have intense ability of migration in this period. Differentiated melanocytes regularly do not proliferate or migrate in adult epidermis and are under tight control of keratinocytes (11). Cutaneous malignant melanoma originating from the melanocytes of the epidermis, is a highly malignant variant of skin tumours with a very poor prognosis if it starts to form metastases, since melanoma cells are highly resistant to any kind of conventional chemotherapy or irradiation. The results of experiments carried out during the last two decades revealed that malignant transformation of melanocytes is associated with overexpression and/or overactivation of various Ser/Thr-specific protein kinases, e.g. protein kinase C (12), protein kinase A (13) or MAP kinases (14). The importance of these signal transduction molecules in the ethiopathogenesis of melanoma is underlined by the fact that pharmacological inhibitors of these enzymes are promising therapeutic drugs of this malignancy (15-17). Although the presumed role of Ser/Thr-protein phosphatases in the altered signal transduction pathways is beyond question,

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little is known about the function of these enzymes in the cellular mechanisms of melanoma cells. As changes of the intracellular Ca-concentration are reported to be involved in the different cellular functions of melanoma cells such as migration (18) or cellular viability (19), it seems likely that calcineurin may play a role in the regulation of these processes. The aim of our study was to investigate the possible role(s) of calcineurin in the major cellular functions of two human melanoma cell lines; one of which (HT168) represents an experimental model of a highly metastatic melanoma (20), while the other (WM35) was isolated from a radial growth phase (RGP) primary lesion (21), and might be a good model of a less malignant, non-metastatic type of this malignancy. We report herein that inhibition of calcineurin by CsA caused different alterations in the morphology, actin organization, proliferation, viability, as well as fibronectin guided migration of the two melanoma cell lines. Furthermore, we found that CsA increased both the expression and the phosphorylation of ERK1/2, and it also elevated the protein level of Bcl-2 and modulated the ß3 and ß1 integrin expression pattern of melanoma cells. Based on our findings we suggest a multiple role of calcineurin in melanoma cells which partly depends on the stage of malignancy. Materials and methods Cultures of melanoma cell lines. Human melanoma cell line HT168 was selected from A2058 cell line according to its metastasis formation in immunosuppressed mice (20), while WM35 was established from a primary cutaneous melanoma of radial growth phase (21). Both cell lines were provided by Dr J. Tímár (National Institute of Oncology, Budapest, Hungary). Cells were cultured in RPMI culture medium (Sigma, Budapest, Hungary) supplemented with 5% foetal bovine serum (HyClone, South Logan, UT), 2 mM L-glutamine (Gibco, Gaithersburg, MD), penicillin (100 units/ml) and streptomycin (100 μg/ml) at 37˚C in the presence of 95% air and 5% CO2 atmosphere and 80% humidity in 25 cm2 flasks (Orange Scientifique, Braine-l'Alleud, Belgium) until approximately 70% confluence. Activity of calcineurin was inhibited by continuous application of 2 μM CsA (Sigma) starting 2 days before confluence.

in a polyclonal Pan-calcineurin A antibody (Cell Signaling Technology, Danvers, MA) at a dilution of 1:400 at 4˚C overnight. For visualisation of the primary antibody, FITCconjugated anti-rabbit secondary antibody (Vector Laboratories, Peterborough, UK) was used at a dilution of 1:1000. Cultures were mounted in Vectashield Hard Set mounting medium (Vector Laboratories) containing DAPI to visualise the nuclei. To demonstrate actin, cells were fixed in acetone at -20˚C for 20 min. After washing at room temperature with calcium magnesium-free phosphate buffer solution (CMFPBS), TRITC-Phalloidin (Sigma) was applied at a dilution of 1:300 (23). Cultures were viewed by fluorescence microscopy (Nikon Eclipse E800, Nikon Corporation). All images were acquired using constant camera settings to allow comparison of staining intensities. Migration assay. Cells were washed twice in CMF-PBS, harvested with 0.25% trypsin (Sigma) and resuspended in RPMI in a density of 2x105 cells/ml. Lower wells of Boyden chamber (Neuro Probe Inc., Gaithersburg, MD) were filled with 1 μl/ml human fibronectin (Sigma) dissolved in CMFPBS and covered with a polycarbonate filter (Neuro Probe Inc.) containing pores with a diameter of 3 μm. Cell suspension (50 μl) was inoculated into the wells on the top of the membrane and the chamber was incubated for 3 h at 37˚C in a humidified atmosphere (5% CO 2-95% air). Non-migrated cells were removed from the surface of the membrane and after fixation in methanol, migrated cells were stained with 1% toluidine blue (Sigma) dissolved in water (24). Membranes were air-dried and mounted with gum arabic. Absolute cell numbers were counted using a light microscope.

Light microscopical morphology analysis. Melanoma cells of different experimental groups were cultured on the surface of rectangular coverglasses (Menzel-Gläser, Menzel GmbH, Braunschweig, Germany) placed into Petri dishes. After rinsing, cells were fixed in a 4:1 mixture of absolute ethanol and 40% formaldehyde, stained with haematoxylin and eosin (Sigma) and mounted in gum arabic. Photomicrographs of cells were taken using a Spot Advanced camera on a Nikon Eclipse E800 microscope (Nikon Corporation, Tokyo, Japan).

Measurement of apoptosis using flow cytometry. After 48 h of treatment with CsA, the amount of apoptotic cells was determined using an Annexin V DY647 kit (Central European Biosystems, Budapest, Hungary) (25) and the ratio of necrotic cells was measured after staining with propidium iodide (PI, Sigma). Untreated cells were used as control. After washing twice in CMF-PBS, cells were incubated in 10 μl Annexin V DY647 and/or PI at room temperature for 10 min. Before harvesting with 0.25% trypsin, cells were washed with Annexin V binding buffer or CMF-PBS. Cell pellets were resuspended in 1000 μl FACS buffer (PBS supplemented with 1% BSA and 0.05% NaN3) and were analysed with a CyFlow® space Flow Cytometer (Partec GmbH, Münster, Germany). Annexin V DY647 was monitored at 670 nm and PI was detected at 430 nm. Measurement lower threshold was set on cell-size particles. Analysis was performed using WinMDI 2.8 freeware (Joseph Trotter, http://facs.scripps.edu/). Quadrants of Annexin/PI density plots were set as follows: Annexin-/PI-; Annexin+/PI-; Annexin-/PI+; Annexin+/PI+ populations.

Immunocytochemistry. For immunocytochemistry cells were cultured on the surface of rectangular coverglasses as described above. For calcineurin immunocytochemistry cells were fixed in Sainte-Marie solution (22) and washed in 70% ethanol. After rinsing in PBS, cells were blocked with PBS supplemented with 1% bovine serum albumin, then incubated

Measurement of cell proliferation with 3 H-thymidine labelling and mitochondrial activity with MTT-assay. Medium containing 1 μCi/ml 3H-thymidine (185 GBq/mM 3H-thymidine, Amersham Biosciences, Budapest, Hungary) was added to cells cultured in wells of 24-well plates for 16 h on 2nd day of CsA treatment. After washing with PBS,

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proteins were precipitated with ice-cold 5% trichloroacetic acid for 20 min. After washing with PBS again, cells were harvested using 0.25% trypsin for 30 min and collected with centrifugation at 2000 x g. The pellet was resuspended in 10 μl CMF-PBS and placed into wells of special opaque 96-well plates (Wallac, PerkinElmer Life and Analytical Sciences, Shelton, CT). The plates were placed in an exsiccator containing phosphorous pentoxide in order to absorb moisture. Prior to measurements, 50 μl scintillation solution (MaxiLight; Hidex Ltd., Turku, Finland) was added to each well and radioactivity was counted by a liquid scintillation counter (Chameleon Microplate Reader, Hidex Ltd., Turku, Finland). For investigation of mitochondrial activity, cells were cultured in wells of 24-well plates and MTTassays were performed measuring the absorption at 570 nm (Chameleon Microplate Reader). Determination of cytosolic free Ca 2+ concentration. Measurements were performed on melanoma cell cultures with approximately 70% confluence using the calciumdependent fluorescent dye Fura-2 (TEFLabs, Inc. Austin, TX). Cell lines were transferred to 2 ml fresh RPMI medium containing 10 ml Fura-2-AM (10 μM) and 4 μl neostigmin (0.3 nM) to inhibit extracellular choline esterases. After 60 min of incubation at 37˚C in a CO2 incubator, cultures were washed twice in Tyrode's solution (137 mM NaCl, 5.4 mM KCl, 0.5 mM MgCl 2, 1.8 mM CaCl 2, 11.8 mM Hepes, 1 g/l glucose, pH 7.4). Fura-2-loaded cells were placed on the stage of an inverted fluorescent microscope and viewed using a x40 oil immersion objective. Measurements were carried out in the above described Tyode's solution in a perfusion chamber using a dual wavelength monochromator (DeltaScan, Photon Technologies International, Lawrenceville, KY) equipment. Fluorescence was measured using excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm. Cytosolic free Ca2+ concentrations were calculated from the ratios of intensities (R = F340/F380) as described (26). Western blot analysis. Total cell lysates were examined by Western blot. Samples for SDS-PAGE were prepared by the addition of 100 μl of 5-fold concentrated electrophoresis sample buffer (20 mM Tris-HCl pH 7.4, 0.01% bromophenol blue dissolved in 10% SDS, 100 mM ß-mercaptoethanol) to cell lysates and boiled for 10 min. About 60 μg of protein was separated by 7.5% SDS-PAGE gel for detection of ERK1/2, p-ERK1/2, calcineurin, Bcl2, integrin ß1, and integrin ß3. Proteins were transferred electrophoretically to nitrocellulose membranes. After blocking in 5% non-fat dry milk in PBST (phosphate buffered saline with 0.1% Tween-20, 20 mM Na2HPO4, 115 mM NaCl; pH 7.4), membranes were washed and exposed to the primary antibodies overnight at 4˚C. Polyclonal anti-ERK1/2 antibody (Sigma) in 1:750, monoclonal anti-diphosphorylated-ERK1/2 (pERK1/2) antibody (Sigma) in 1:400, polyclonal anti-PP2B (Pan-calcineurin A) antibody (Cell Signaling Technology, Danvers, MA) in 1:400, monoclonal anti-Bcl2 antibody (Upstate, Dundee, Scotland, UK) in 1:500, monoclonal antiintegrin ß1 antibody (Chemicon-Millipore, Billerica, MA) in 1:400 and polyclonal anti-integrin ß3 antibody (Cell

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Signaling Technology, Danvers, MA) in 1:400 dilution were used. After washing for 30 min with PBST, membranes were incubated with secondary antibodies, anti-rabbit IgG (Bio-Rad Laboratories, CA) in 1:1000 dilution for ERK1/2, calcineurin and integrin ß3, anti-mouse IgG (Bio-Rad Laboratories) for Bcl2, p-ERK1/2 and integrin ß1 in PBS containing 1% non-fat dry milk for 1 h at room temperature. Signals were detected by enhanced chemiluminescence (Pierce, Rockford, IL) according to the instruction of the manufacturer. RT-PCR analysis. For RT-PCR analysis, melanoma cells were washed three times with RNase-free physiological NaCl, then the cultures were snap-frozen in liquid nitrogen and stored at -70˚C. Cell cultures were dissolved in TRIzol (Applied Biosystems, Foster City, CA), and following addition of 20% RNase free chloroform (Sigma) samples were centrifuged at 10,000 x g for 15 min at 4˚C. Samples were incubated in 500 μl RNase free isopropanol at -20˚C for 1 h, total RNA was dissolved in RNase free water and stored at -70˚C. The assay mixture for reverse transcriptase reactions contained 2 μg RNA, 0.112 μM oligo(dT), 0.5 mM dNTP and 200 units of High Capacity RT (Applied Biosystems) in 1X RT buffer. The sequences of primer pairs for polymerase chain reaction were as follows: for calcineurin 5'-TAC CCT GCA GTT TGT GAA TT-3' and 5'-ATA TGT TGA GCA CAT TTA CCA-3', for ERK1/2 5'-CCA GAC CAT GAT CAC ACA GG-3' and 5'-CTC GTC ACT CGG GTC GTA AT-3', for Bcl2 5'-GGG TAC GAT AAC CGG GAG ATA-3' and 5'-GGC CGT ACA GTT CCA AG-3', for integrin ß1 5'-GCC TAC TTC TGC ACG ATG-3' and 5'TAA ATG TCT GTG GCT CCC-3', for integrin ß3 5'-CGT CCT GAC GCT AAC TGA-3' and 5'-GGT AGT GGA GGC AGA GTA ATG-3', for GAPDH 5'-CCA GAA GAC TGT GGA TGG CC-3' and 5'-CTG TAG CCA AAT TCG TTG TC-3'. At defined annelation degrees 30 cycles were used and PCR products were analysed by electrophoresis in 1.2% agarose gel containing ethidium bromide (Amresco Inc., Solon, OH) and photographed with a gel documentary system (DNR Bio-Imaging Systems Ltd., Jerusalem, Israel). Calcineurin activity assay. For calcineurin activity assays, cells were harvested and after centrifugation at 10,000 x g for 10 min at 4˚C, the supernatants were used for enzyme activity measurements. Activity of calcineurin was assayed by the release of 32Pi from 32P-labelled protein phosphatase inhibitor-1 as it was described by Yang et al (27) with some modifications (28). Radioactivity was determined by Cerenkov counting in a liquid scintillation counter. Statistical analysis. Statistical comparisons between control and test samples were analyzed using Student's paired t-test for the cell migration, mitochondrial activity and proliferation assays. Results Both the expression and the activity of calcineurin are higher in the more malignant HT168 cells. As there are no data either on the expression or the enzymatic activity of calcineurin

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Figure 1. Detection of calcineurin in two different melanoma cell lines. (a) mRNA expression of calcineurin in HT168 and WM35 cells before and after the application of 2 μM CsA. GAPDH was used as a control. Representative data of five independent experiments. (b) Protein expression level of calcineurin in untreated control cells and under the effect of 2 μM CsA. Representative measurement of five independent experiments. (c-f) Subcellular localization of the catalytic subunit of calcineurin by immunochemistry. Untreated cells of HT168 (c), HT168 cells treated with 2 μM CsA (d), untreated WM35 cells (e), WM35 cells treated with 2 μM CsA (f). Original magnification was x40. Representative photomicrographs of four independent experiments. (g) Enzymatic activity of calcineurin. Asterisks indicate significant (*P